Biomass immobilization
TMM containing HTN biomass grown at logarithmic phase (1.87 × 1010 CFU) was fed into the reactor via a peristaltic pump with a liquid recirculation and air flow rates of 3.6 L/h and 0.5 L/min. These flow rates were fixed throughout experiments. During the immobilization process, three strategies were used to optimize the cell attachment and sulfur oxidation regarding the recirculating medium as following; (1) fresh TMM medium was replaced when pH dropped lower than pH 4.5; (2) there was no replacement of fresh TMM medium after inoculation but pH was adjusted to 7 every 24 h; (3) fresh TMM medium was replaced every 48 h and pH was adjusted to 7 every 24 h. Abiotic control was tested by using TMM broth without HTN inoculation. The reactor was operated under room temperature (30–32 °C). The attached packing media was weighted following each of the experiments after the system reached steady state with respect to sulfate production. The growth of microorganism was determined by calculation of the difference of weight between before and after process. The recirculating medium was sampled periodically to determine sulfate concentration, colony forming unit (CFU/mL), and pH.